The purpose of this study is to probe the mechanism by which cytochrome P-450a converts testosterone to the metabolite I7beta- hydroxy-4,6-androstadiene-3-one (delta 6T). Previous work in this Laboratory, which showed that 6T formation paralleled 6-beta- hydroxylation, suggested that the 6 beta-hydroxylate catalyzed the reaction and led to the concept of a dual hydrogen atom abstraction mechanism. In addition, however, desaturation reaction is also catalyzed by cytochrome P-450a, which hydroxylates testosterone preferentially to 7 alpha-hydroxytestosterone. An analogous metabolite of valproate has also been reported. The objective of this work is to measure the deuterium isotope effects associated with the desaturation mechanism. Selectively deuterated testosterone derivatives were synthesized and subjected to metabolism by reconstituted systems of cytochrome p-450a. The isotope effects for delta 6T formation strongly support a stepwise mechanism in which the 6 alpha-hydrogen atom is initially abstracted, followed 7 alpha-hydrogen atom abstraction to generate the unsaturated metabolite and water.